Project Summary Roles of antibodies in AIDS are poorly understood, in part because cell culture definitions of neutralization are uncertain. Although structures of gp120-gp41 complexes with neutralizing antibodies are known, the mechanism(s) by which they lower HIV-1 titers are unproven. Nevertheless, it has been assumed that neutralizing antibodies directly and perhaps irreversibly inactivate HIV-1. We made unique resources (e.g., cell clones with discrete amounts of CD4 and CCR5 including JC.53 cells used to make the TZM-bl derivative) that are widely used for vaccine and entry inhibitor analyses. We also isolated adapted HIV-1 variants that efficiently use damaged CCR5s [e.g., CCR5([unreadable]18) lacking the tyrosine-sulfated amino terminus (Nt) and others that efficiently use CCR5([unreadable]18) only while Nt is present in the medium]. Recently, by observing HIV-1 virions adsorbed onto JC.53 cells, we found that infectivity is substantially determined by a race between entry and a previously undescribed process of dissociation. Polycations enhance infectivity not by increasing attachment as previously supposed but by preventing dissociation. Consistent with this kinetic competition model of infectivity, CCR5 antagonists slow entry and their inhibitory potencies are reduced by polycations. Unexpectedly, the 2G12 monoclonal antibody (MAb) also reduces HIV-1 titers by slowing entry. Thus, neutralization results indirectly from process(es) that kinetically compete with infection in the assay conditions, and single virions are increasingly slowed but not irreversibly inactivated when 2G12 binding increases. We propose to learn whether other MAbs classified as neutralizing function kinetically or by alternative means, and to evaluate the in vivo relevance of different neutralization mechanisms. We propose: (i) Determine whether MAbs classified as neutralizing generally slow entry or whether some directly inactivate virions. Learn how redundancy of envelope glycoproteins on virions contributes to entry speed and resistance to neutralization. Study the relevance of standard and novel HIV-1 neutralization assays to in vivo conditions by analyzing available neutralizing antibodies and escape mutations in SHIVSF162 gp120-gp41 that coevolved in infected macaques. (ii) Dissect roles of CCR5 regions in mediating steps of HIV-1 entry. (a) Isolate adapted HIV-1s that are increasingly reliant on Nt, with the goal of ultimately obtaining variants that can use soluble Nt as coreceptor. (b) Using HIV-1s that efficiently infect HeLa-CD4/CCR5([unreadable]18) cells or T-lymphocyte/CCR5([unreadable]18) cells only while Nt is present, synchronously induce or block entry of stably adsorbed virions in physiological conditions. Learn which intermediates are sensitive to specific inhibitors and antibodies. Because Nt is membrane impermeable, it provides a powerful means to evaluate the role of endocytosis in HIV-1 entry and in escape from antibodies. This project uses unique resources and approaches to understand kinetics of HIV-1 infection, mechanisms of viral adaptation to entry limitations including neutralizing Abs, and related issues important for optimization of entry inhibitors and vaccines for AIDS.